One of the most interesting aspects of modern cell biology is the mechanism by which cells and tissues respond to various kinds of stress. For the heart this response is especially important, because the changing demands induce a protection which is essential for survival. A temporary stimulation of the heart is regulated by changes in diastolic volume (preload), aortic pressure (afterload), heart rate, adrenergic mechanism, and substrate availability. If the increase in demand is chronic the response leads to the development of adaptional factors. Two processes are of importance: i) hypertrophy due to an increase in the size of the myocytes and ii) an elevated efficiency of the contraction of each sarcomere. The restructuring of the different components of the cell depends on the nature, duration, and intensity of the stress, as well as on the age and the species (N. R. Alpert, L. A. Mulieri, Med. Sci. Sports Exerc., 18, 309-313 (1986)). This is particularly true for muscle cells. Muscles are classified broadly into the two groups of striated muscles and smooth muscles. Striated muscles are further classified into cardiac muscles and skeletal muscles, the skeletal muscles being further classified into fast muscles and slow muscles. It has been reported that these can be distinguished immunochemically through the difference in immunogenicity of the myosin molecules which are major constituents of muscles (Masaki et al., J. Biochem., 76, 441, (1974)). At least two molecular variants of myosin heavy chains, coded by distinctive genes (V. Mahdavi, V. A. P. Chambers, B. Nadal-Ginard, Proc. Natl. Acad. Sci. USA., 81, 2626-2630 (1984)), have been described in the human myocardium: an atrial HC.alpha.- and a ventricular HC.beta.-type (L. Gorza, J. J. Mercadier, K. Schwartz, L. E. Thornell, S. Satore, S. Schiaffino, Circ. Res., 54, 694-702 (1984); J. J. Mercadier, P. Bouveret, L. Gorza, S. Schiaffino, W. A. Clark, R. Zak, B. Swynghedauw, J. Schwartz, Circ. Res., 53, 52-63 (1983); H. O. Hirzel, C. R. Tuchschmid, J. Schneider, H. P. Krayenbuehl, M. C. Schaub, Circ. Res., 57, 729-740 (1985); M. Kuro-O, H. Tsuchimochi, Uedas, F. Takaku, Y. Yazaki, J. Clin. Invest., 77, 340-347 (1986); H. Tsuchimochi, M. Kuro-O, F. Takaku, K. Yoshida, M. Kawana, S. Kimata, Y. Yazaki, Jap. Circ. J., 50, 1044-1052 (1986); C. Dechesne et al., J. Mol. Cell. Cardiol., 17, 753-767 (1985); P. Bouvagnet et al., Circ. Res., 55, 794-804 (1984); H. Tsuchimochi et al., J. Clin. Invest. 81, 110-118, (1988)).
They differ in both ATPase activity and mobility in pyrophosphate polyacrylamide gel electrophoresis. HC.alpha. has a higher Ca.sup.++ - and actin-activated ATPase activity than does HC.beta. and migrates faster in gels (Yasaki et al., Circ. Res., 35, 15, (1974); Hoh et al., J. Mol. Cell. Cardiol., 10 1053-1076, (1978)).
These two types of heavy chains have been shown in animals to form myosin molecules composed either of an .alpha..alpha. homodimer or an .alpha..beta.-heterodimer, or a .beta..beta.-homodimer which correspond to the V-1, V-2 and V-3 isoforms described by Hoh and coworkers (J. F. Y. Hoh, J. Mol. Cell Cardiol., 10, 1053-1076, (1978)). V-1 exhibits higher adenosine triphosphatase (ATPase) activity than V-3.
The ratio of these myosin isoforms varies according to the physiological and pathological state or developmental stage of the myocardium (for reviews, see Mercadier et al., Circ. Res., 53, 52-62, (1983); Tobacman et al, J. Biol. Chem., 259, 11226-11230, (1984)). The change from V-1 toward V-3 is accompanied by a decrease in ATPase activity and speed of contraction (Schwartz et al., J. Mol. Cell Cardiol., 13, 1071-1078, (1981); Ebrecht et al., Basic Res. Cardiol., 77, 220-234, (1982)), an improved economy of force generation (Alpert et al., Fed. Proc. 41, 192-198, (1981); Alpert et al., Circ. Res., 50, 491-500, (1982)), and decreased oxygen consumption (Kissling et al., Basic Res. Cardiol., 77, 255-270, (1982)). These changes in myosin HC composition found in animals were interpreted as an adaptation of the myocardial cell, together with compensatory hypertrophy of the muscle, to new functional requirements.
In man, normal ventricular tissue contains predominantly the V-3 species (.beta..beta.-homodimer) and only few amounts (0-15%) of the V-1 species (.alpha..alpha.-homodimer). The opposite is true for the human atrium, where the abundant myosin isoform is V-1 and, to a lesser degree, isozyme V-3.
Human fetal atrium is composed mostly of .alpha.-HC during the first 23 weeks of gestation. (Bouvagnet et al., Circ. Res., 61, 329-336, (1987)). .beta.-HC is already expressed as traces at 14 weeks of gestation, and its expression increases progressively until birth, resulting in a great augmentation in .beta.-HC. During this course, .beta.-HC always predominates in certain areas (the crista terminalis and the interatrial septum) but not in other areas (the auricles). Preceding birth, the fetal ventricle is composed mostly of .beta.-HC. From 14 weeks of gestation to birth, .alpha.-HC is expressed in very rare fibers. Then, after birth, a large number of fibers simultaneously synthesize .alpha.-HC.
Animal studies have shown evidence of thyroid hormone affecting the expression of isomyosin (for review, see B. Swynghedauw, Physiol. Rev., 66, 710-771, (1986)). It is not yet known whether the human heart has the same property, but at birth, increase of .beta.-HC expression in the atrium and .alpha.-HC in the ventricle, is associated with the rapid rise in circulating thyroid hormone levels. Since Chizzonite and Zak (J. Biol. Chem., 259, 12628-12632, (1984)) have clearly demonstrated the role of thyroid hormone in the induction of .alpha.-HC expression in neonatal rat ventricle, thyroid hormone hypothetically could also induce ventricular .alpha.-HC expression in humans. Thyroid hormone has highly tissue specific effect and can switch HC gene expression on or off depending on the tissue where it is expressed (Izumo, et al., Science, 231, 597-600, (1986)). Insulin also is involved in the regulation of myosin HC expression (Dillman, et al., J. Biol. Chem., 259, 2035-2038, (1984)).
In pressure-overloaded human atrial muscle, there is a transition from .alpha.- to .beta.-HC (Kurabayashi, et al., J. Clin. Invest., 82, 524-531, (1988); Tsuchimochi, et al., J. Clin. Invest., 74, 662-665, (1984); Buttrick, et al., Circulation, 74, 477-483, (1986); Schlesinger, et al., Biochem. Intern. 11, 747-753, (1985)) as an early adaptation to the imposed load.
Myocardial infarction and associated work overload cause a transition in the light chain complements of the myosin (Hoffman, et al., Basic Res. Cardiol. 32, 359-369, (1987); Hoffman et al., Biomed. Biochem. Acta, 46, S724-S727, (1987)). Ventricular myosin light chains are found in pressure overloaded atria and atrial light chains have also been identified in the infarcted ventricle of the human heart. The relative proportions of atrial myosin heavy chains are changed after infarction. A decrease in .beta.-HC and a corresponding increase in .alpha.-HC were observed. Ventricular hypertrophy in patients with coronary insufficiency induces .alpha.-HC expression. The relative part of this myosin type amounts to 20%.
In the field of muscle research, antibodies against muscle proteins have long been utilized.
In recent years, as a method for obtaining large amounts of an antibody having high specificity, it has been known to prepare a hybridoma, by fusion of an antibody-producing cell with a myeloma cell and culturing the hybridoma thus obtained to produce a monoclonal antibody (Kohler et al., Nature, 256, 495 (1975)), and a large number of monoclonal antibodies have been obtained by such a method.
Further, these antibodies can be labeled with radioisotopes and used for localization of myocardial infarction.
There is provided in U.S. Pat. No. 4,767,843 (Yazaki et al.) a monoclonal antibody which has specificity to cardiac myosin heavy chain .alpha. type but does not recognize cardiac myosin heavy chain .beta. type and also a monoclonal antibody which has specificity to cardiac myosin heavy chain .beta. type but does not recognize cardiac myosin heavy chain .alpha. type.
Although the fate of myosin following myocardial infarction is not exactly known, the published experimental evidence, allows one to expect that certain fragments of myosin remain in situ in myocyte while others are released to circulation. The classic light microscopic studies of the heart after myocardial infarction in man demonstrated that disappearance of muscle fibers is relatively slow, beginning about 6 hr after infarction and continuing for 2 to 6 weeks. While it has been reported that myosin with an intact ATPase activity can be extracted from infarcted muscle for as long as one month after acute infarction it has also been noted that there is a marked decrease in the amount of myosin in the infarcted zone within 24 to 48 hr of the infarct.
Among the potential causes of ischemia-induced cell damage is the acute intracellular acidosis that occurs after coronary artery occlusion. The decreased contractility of acutely ischemic myocardium and a decrease in actomyosin ATPase activity are the consequences of an acidosis.
Therefore, acidification of myosin due to ischemia could cause dissociation of light chains of myosin from heavy chains, and fragmentation of heavy chains, suggesting a possible source of circulating light chains and fragments of heavy chains after myocardial infarction. Ongoing release of myosin fragments could also be explained by proteolytic degradation of myofibrils by such enzymes as Ca.sup.2+ -activated proteinases, cathepsins B and D, and possibly cathepsins H and L. Thus, following myocardial infarction the free myosin is degraded by proteolytic enzymes or dissociated by the pH shift liberating more myosin fragments into circulation where they remain up to the 10-14th day after the onset of chest pain.
Several immunoassays for the quantitation of cardiac myosin light and heavy chain fragments in sera of patients with acute myocardial infarction (AMI) have been described and found useful in the diagnosis of AMI and estimation of infarct size (Katus et al., Am. J. Cardiol., 54:964-970 (1984); Hirayama et al., Clin. Biochem., 23:515-522 (1990); Leger et al., Eur. J. Clin. Invest., 15:422-429 (1985); Larue et al., Clin. Chem., 37: 78-82 (1991); Leger et al., Am. Heart J., 120:781-790 (1990)).
To detect circulating myosin heavy chain fragments, Leger et al. used murine monoclonal antibodies (MAbs) to the .beta.-type myosin HC of human ventricle (Leger et al., Eur. J. Clin. Invest., 15:422-429 (1985)). However, not every anti-.beta. myosin HC antibody is suitable for the recognition of circulating myosin HC, and thus for the measurement of myosin fragments in patients' plasma. Because in the circulation myosin is effectively proteolyzed into fragments as soon as it is liberated from the myocyte, only monoclonal antibodies that recognize those myosin fragments most resistant to proteolysis can be used in an assay. Chemotryptic subfragment 2 of the rod of heavy meromyosin (HMM) owing to the central position is very resistant to proteolysis. Thus, antibodies recognizing epitopes on the subfragment 2 of heavy meromyosin should be the best candidates for an assay detecting serum myosin fragments (Larue et al., Clin. Chem., 37: 78-82 (1991)).
On the contrary, antibodies directed to the subfragment 2 of heavy meromyosin are not the desirable candidates of in vivo imaging of AMI since they would cross-react with plasma myosin, causing high blood pool radioactive background. The only candidates for in vivo imaging are those antibodies that bind to myosin heavy chains that remain in situ after myocyte death and membrane disintegration, and only those that react with epitopes common to .alpha. and .beta.-types of myosin HC.
Proteolytic enzymes dissociate myosin to light (LMM) and heavy (HMM) meromyosin fragments which can be further fragmented to subfragments 1 (SF1) and 2 (SF2) (Margossian and Lowey, Methods in Enzymol., 85:55-71 (1982)). LMM fragment of the rod is non-soluble in physiological fluids and is responsible for thick filament formation. The .alpha. and .beta. MHC nucleotide and amino acid sequences are 97% identical in this region (Mahdavi et al., Proc. Natl. Acad. Sci. USA, 81:2626-2630 (1984); McNally et al., J. Mol. Biol., 210:665-671 (1989)).
In view of the above presented evidence, it would be highly desirable to have a monoclonal antibody which recognizes .alpha.- and .beta.-heavy chain of atrial and ventricular human myosin for imaging of myocardial infarction, since a single antibody molecule would demarcate the infarcted zone regardless of the patient's age, the pathological or the physiological condition.
Thus, it would be highly desirable to be provided with antibodies directed to LMM fragment of cardiac myosin which may be the ideal candidates for AMI imaging.
Further, it would also be useful to have a monoclonal antibody which could provide the diagnosis of both atrial and ventricular myocardial infarction simultaneously.